Determination of endometrial receptivity toward embryo implantation

ABSTRACT

Methods for detecting receptivity of mammalian endometrium to embryo implantation comprising obtaining a sample of the endometrium, contacting the endometrium with a monoclonal antibody for β 3  and detecting β 3  in the endometrium. The invention also provides for methods of diagnosing infertility in a mammal and methods of detecting the window of embryo implantation in endometrium. Methods of in vitro fertilization, methods of preventing embryo implantation and a method of monitoring endometrial maturation are also within the scope of the present invention. The present invention is also directed to contraceptives. Diagnostic kits useful in the practice of the methods of the invention are also provided.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/149,183, filed Sep. 8, 1998, which is a continuation of U.S.application Ser. No. 08/756,211. This application also is a continuationof U.S. application Ser. No. 08/756,211, filed Nov. 25, 1996, which is adivisional of U.S. application Ser. No. 08/400,270, filed Mar. 3, 1995,now U.S. Pat. No. 5,578,306, which is a continuation of U.S. applicationSer. No. 08/126,063, filed Nov. 1, 1993, now abandoned, which is adivisional of U.S. application Ser. No. 08/897,706, filed Jun. 12, 1992,now U.S. Pat. No. 5,279,941. Benefit of priority under 35 U.S.C. §120 toeach of the above-noted applications is claimed.

REFERENCE TO GOVERNMENT GRANTS

[0002] This work was supported in part by research grants from theBiomedical Research Support Grant Program, Division of ResearchResources, the National Institutes of Health, grant number B.R.S.B.S07-RR-0541 5-29. The United States Government may have certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] Over the past decade, investigators have come to recognize theimportance of the extracellular matrix (ECM) in directing the growth,differentiation and function of the overlying epithelium. Getzenberg etal., “The Tissue Matrix: Cell Dynamics and Hormone Action”, EndocrineRev., 11:399-417 (1990). The interaction between cell and extracellularmatrix (or substratum) is mediated by several classes of cell adhesionmolecules, one of the most important being the integrins. Albelda etal., “Integrins and Other Cell Adhesion Molecules”, FESEB J.,4:2868-2880 (1990). Buck et al. “Integrin, a Transmembrane GlycoproteinComplex Mediating Cell-Substratum Adhesion”, J. Cell Sci. Suppl.,8:231-250 (1987). This diverse family of glycoprotein receptors isexpressed on the cell membrane as heterodimeric α and β subunits and isinvolved in both cell-cell and cell-substratum adhesion. Specificrecognition and binding of extracellular matrix (ECM) components such asfibronectin (FN), laminin (LM) and collagen (Col) transmit informationto the cytoskeletal structure, an interaction which may have major rolesin promoting hormone responsiveness and genomic activation. Burridge etal., “Focal Adhesions: Transmembrane Junctions Between the ExtracellularMatrix and the Cytoskeleton”, Ann. Rev. Cell. Biol. 4:487-525 (1988) andGetzenberg et al. supra.

[0004] Although extensive information exists about specific integrinproteins, for example, Hemler, M. E. “VLA Proteins in the IntegrinFamily: Structures, Functions and Their Role on Leukocytes”, Annu. Rev.Immunol: 365-400 (1990), little is known concerning the distribution ofthese receptors in the female reproductive tract. In the uterus, theendometrium, composed of glandular epithelium and associated mesenchyme(stroma), maintains complex temporal and spatial functions in responseto the cyclic hormonal milieu. The search for morphological orbiochemical markers for uterine receptivity has been unsuccessful todate as reported by Rogers and Murphy, “Uterine Receptivity forImplantation: Human Studies”, in Blastocyst Implantation, Yoshinaga, K.ed., Serono Symposia, pp. 231-238 (1989). Once such markers areidentified, their role in endometrial phenomena including embryoimplantation, fertility, contraception and endometrial maturation andreceptivity can likely also be identified. Thus, as some integrinsappear to meet the criteria for markers of receptivity there is a greatneed for methods of detecting integrin cell adhesion molecules inendometrium.

SUMMARY OF THE INVENTION

[0005] The present invention is directed to methods of detectingreceptivity of endometrium to embryo implantation by detecting the β₃subunit of the α_(v)/β₃ integrin in endometrium with a monoclonalantibody.

[0006] Methods of diagnosing fertility and methods of monitoringendometrial maturation in a mammal are also provided by monitoring theappearance of the β₃ subunit of integrin in endometrium from a pluralityof stages of the endometrial cycle. This is preferably done with amonoclonal antibody.

[0007] The present invention also provides methods of detecting theoptimal window of embryo implantation in the endometrium by detectingthe β₃ subunit of integrin in an endometrial sample, preferably with amonoclonal antibody.

[0008] Further aspects of the invention include methods of preventingembryo implantation by contacting the β₃ subunit of integrin in theendometrium with neutralizing Fab antibody fragments to β₃. Methods ofin vitro fertilization are also embodiments of the invention. Thesecomprise detecting the β₃ subunit of integrin in an endometrial sample,fertilizing an egg in vitro, and introducing the zygote into the uterushaving endometrial tissue expressing the β₃ subunit.

[0009] Contraceptive and diagnostic kits are also contemplated hereby.

[0010] These and other aspects of the invention will become moreapparent from the following detailed description when taken inconjunction with the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 depicts immunoperoxidase staining of normal endometrium.The photomicrographs depict the pattern of distribution for sixdifferent integrins that do not appear to change throughout the menstralcycle. Dark areas represent positive staining, light areas representabsence of stain (absence of specific integrin subunit).Immunohistochemical staining of the collagen/laminin receptor subunits:α₂(A), α₃(B), α₆(C), and β₄(D) shows prominent staining of epithelium(←) and microvessels (←) without significant stromal staining (*) forα₂, α₃, and β₄. Note basolateral staining α₆ and basal staining for β₄.Staining for fibronectin receptor subunits α₄(E), α₅(F) show predominantmesenchyme staining (*) with decreased epithelial staining (←). Theimmunoreactions (areas of dark staining) were developed byavidin-biotin-peroxidase complex using diaminobenzidine as a chromogen.For greater sensitivity, no counterstain was applied.

[0012] Magnification: 125×.

[0013]FIG. 2 shows photomicrographs of the immunohistochemical stainingfor the integrin subunit α₁ in proliferative vs. secretory endometrium.The staining in the glandular epithelium (←) was largely absent in theproliferative phase (A), and pronounced in all sections after menstrualcycle day 14 (B; day 20 endometrium). The microvasculature (←) stainingwas also pronounced, and did not change throughout the menstrual cycle.The staining noted in secretory endometrial glands was significantlyhigher than that of background (C).

[0014] Magnification: 125×.

[0015]FIG. 3 exhibits immunostaining of α_(v) and β₃ (the two pairingsubunits of the vitronectin receptor integrin) in proliferative phasevs. secretory phase endometrium. The staining intensity of α_(v) in theproliferative phase (A) was judged as “+” for the stromal cells (*) and“±” for glandular α_(v) (←). Immunostaining for a α_(v) in day 22endometrium (B) demonstrates a significant increase in glandularstaining (example of “++” staining intensity). Likewise, the stainingfor β₃ was absent in proliferative epithelium (C; ←) and was notablyincreased in this day 22 secretory endometrium (D). Magnification: 125×.

[0016]FIG. 4 shows relative intensity of staining for the epithelialα_(v) and β₃ subunits in 35 endometrial samples throughout the menstrualcycle. The pattern of expression for β_(v) is shown in A, shows agradual increase in staining throughout the menstrual cycle. Incontrast, the pattern for β₃ in B, shows a more abrupt rise in thisintegrin subunit around day 20 of the menstrual cycle. Samples werestaged according to the last menstrual cycle. Sections were assigned ascore of 0 (−; negative), 1 (±; weak), 2 (+; moderate) or 3 (++;strong), by a blinded observer, and confirmed by a second observer.

[0017]FIG. 5 depicts staining intensity of epithelial β₃ in 12infertility patients with delayed endometrial maturation. Endometriumwas collected from women undergoing evaluation for infertility. Thebiopsies were separated into two groups based on the correlation betweenhistologic criteria and the menstrual cycle dating based on the time ofovulation and/or the subsequent menstrual period. Patients withendometrial biopsies 3 or more days “out of phase” (OOP group) werecompared with 25 endometrial biopsies that were “in phase” (Normal) andshown in A. Sections were assigned a score of 0 (−; negative), 1 (±;weak), 2 (+; moderate) or 3 (++; strong), based on the intensity ofepithelial β₃ staining. Examples of immunohistochemical staining of an“out of phase” biopsy (B) and a normal “in phase” sample (C) is includedto contrast the epithelial β₃ staining in each group. Magnification:400×.

[0018]FIG. 6 exhibits immunoblot analysis of proliferative and secretoryendometrium, stained for the β₃ subunit. (A) Immunoblot of plateletextract (lane 1) compared with 2 samples from the early and midproliferative phase (lanes 2,3) and from the luteal phase (lane 4 and 5;days 23 and 26, respectively) demonstrates a band at approximately 95 kDmolecular weight, corresponding to β₃. Samples of endometrium werepartially digested with collagenase and the glandular elements obtained(B) using a modification of the methods of Satyaswaroop et al.,“Isolation and Culture of Human Endometrial Glands”, J. Clin. Endocr.Metab., 48:639-641 (1979). The glands appear as hollow structures freefrom surrounding stroma. Immunofluorescence of samples from lanes 3 and4 (C and D, respectively) corresponds to the absence or presence of the95 kD band in A. Magnification: 400×.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0019] The present invention is directed to methods of detectingreceptivity of mammalian endometrium including obtaining a sample ofendometrium, contacting the sample with a monoclonal antibody for the β₃subunit of integrin and detecting the β₃ subunit.

[0020] For purposes of the present invention, the β₃ subunit may be β₃alone or β₃ in combination with another integrin subunit, α_(v), forexample.

[0021] As used herein integrin is defined as a diverse class ofglycoprotein receptors expressed on the cell membrane. Integrins arecell adhesion molecules of the immunoglobulin superfamily. Integrins arecomposed of heterodimeric α and β subunits and are involved in cell—celland cell—extracellular matrix adhesion. The integrin family is a broadlydistributed group of receptors composed of noncovalently associated α/βheterodimer pairs that mediate leukocyte—leukocyte andleukocyte—endothelial cell adhesion, as well as cellular interactionswith extracellular matrix components such as collagen, laminin,fibrinogen and fibronectin, and cell-cell interaction in organizedtissues.

[0022] While integrins are found on virtually all cell types (theexception being red blood cells), expression of integrin subunits variesfrom cell type to cell type. In human uterine endometrium, as determinedherein, glandular epithelial cells express primarily α₂, α₃ and α₆integrin subunits, which are collagen laminin receptors. Stromal cellsexpress predominantly α₅, a fibronectin receptor. The presence of α₁ onglandular epithelial cells is menstrual cycle specific, found onlyduring the secretory phase. Expression of both subunits of thevitronectin receptor, α_(v)/β₃, also undergoes menstrual cycle specificchanges on endometrial epithelial cells. The expression of α_(v)increases throughout the menstrual cycle while the β₃ subunit appearsabruptly on menstrual cycle day 20 on luminal and glandular epithelialcells.

[0023] The present invention is directed to endometrium of the uterus ofa mammal. The uterine wall is largely smooth muscle or myometrium. Theendometrium, a glandular layer of variable thickness extremely sensitiveto the hormones estrogen/progesterone, lines the myometrium. Theendometrium is composed of several functional layers. The layer nearestthe myometrium is termed the basalis layer, and the layer closer to thesurface known as the functionalis. This tissue is made of epithelialcells, stromal (or mesenchymal) cells and endometrial leukocytes. Theepithelial cells are either glandular (forming glands beneath thesurface of the endometrium) or luminal (lining the surface of theendometrium). These different types of epithelium serve differentpurposes and staining patterns for different marker proteins are notalways the same between glandular and luminal. It is the luminal surfacethat would encounter the human embryo first and is thought to beinvolved in initial attachment. The endometrium of premenopausal girlsand postmenopausal women is atrophic due to the lack of the hormones,estrogen and progesterone. In the reproductive-aged woman, theendometrium undergoes cyclic developmental changes based on the ovariancycle of hormone release. The first day of menstruation is the first dayof the cycle; menstruation is generally completed by day 5. Theendometrial growth then resumes under the influence of estrogen andprogresses through the day 14, proliferative phase, and on to about day28. From day 14 to day 28 the endometrium also shows signs of dncreasedgland growth and secretion, secretory phase, due largely to theinfluence of progesterone. During the follicular phase, while folliclesare growing in the ovary, and estrogen is the dominant hormone, theendometrium grows thicker. With ovulation (typically day 14 of a 28 daycycle) the women is exposed to estrogen plus progesterone. This iscalled the secretory or luteal phase, and is noted for a stereotypicseries of histologic changes that proceeds as the cycle continues. Thesehistologic changes are used by pathologists to date the endometrium, aprocess that remains controversial despite its use for the past 40years. There have been no reliable immunohistochemical markers reportedthat have proven utility in dating the endometrium.

[0024] Luteal phase dysfunction (LPD) is a term for developmental delayof the endometrium. It is a known cause of infertility, because ofdyssynchrony between the fertilized egg and the endometrium. If anembryo is ready to attach but the endometrium is delayed, then pregnancyis not likely to occur. The causes for LPD include inadequate hormonaloutput by the ovary, and may implicate defective signally from highercenters such as inadequate gonadotropic hormone output from thepituitary or hypothalamus. LPD is a known cause of infertility andspontaneous abortion and can be corrected with hormone augmentation.

[0025] Embryo implantation stages include: apposition—when theepithelial cells of the embro attach to the outer (luminal) epithelialcells of the maternal endometrial surface; adhesion; and invasion oftrophoblast into the underlying stroma where it established itself andbegins to grow. Contact with maternal blood vessels is made to gainnutrients and oxygenated blood and to rid itself of waste productsduring the invasion stage. The stage of development that the embryoreaches at the time of implantation is the blastocyst stage, whichoccurs at the same time as hatching. There is evidence that hatching isrequired before implantation occurs, perhaps becuase the embryo musthave its epithelial cells exposed (out of the zona pellucida shell) tointeract with the maternal cell layers. As set forth herein, thisinteraction occurs via integrins.

[0026] For purposes of the current invention, mammals include, but arenot limited to the Order Rodentia, such as mice; Order Logomorpha, suchas rabbits; more particularly the Order Carnivora, including Felines(cats) and Canines (dogs); even more particularly the OrderArtiodactyla, Bovines (cows) and Suines (pigs); and the OrderPerissodactyla, including Equines (horses); and most particularly theOrder Primates, Ceboids and Simoids (monkeys) and Anthropoids (humansand apes). The mammals of most preferred embodiments are humans.

[0027] Monoclonal antibodies useful in the practice of the inventioninclude any monoclonal antibodies having an affinity to or binding tothe β₃ subunit of integrin. An example of such a monoclonal antibody isSSA6. Monoclonal antibody SSA6 may be produced as described by Bennettet al., PNAS, Vol. 80, 2417-2421 (1983).

[0028] Monoclonal antibodies which recognize β₃ combined with anotherintegrin subunit may also be used. One such monoclonal antibody is 23C6,which may be prepared according to the method of Davies et al., J. CellBiol., Vol. 109, 1817-1826 (1989). Immunostaining with monoclonalantibodies such as 23C6 (specific to the intact α_(v)/β₃ integrin, i.e.the vitronectin receptor) produces the identical pattern as SSA6 whichonly measures the β₃ subunit. This demonstrates that while α_(v)specific antibodies measure all the α_(v) containing integrins,antibodies which recognize the intact α_(v)/β₃ integrin or the β₃subunit can be used to study this integrin (the α_(v)/β₃ “vitronectinreceptor”).

[0029] Other monoclonal antibodies can be used. The preparation ofmonoclonal antibodies is known to those in the art. Particularly, themethod of Kohler and Milstein, Nature, 256: 495-497 (1975) may be usedto produce monoclonal antibodies for use in the invention.

[0030] Methods of obtaining endometrial tissue samples for analysis,include any surgical and nonsurgical technique known in the art.Surgical methods include, but are not limited to biopsy, dilation andcurettage. Nonsurgical methods include, but are not limited to, uterinewashings and uterine brushings with immunocytochemical evaluation.

[0031] Methods of detecting β₃ in the endometrium include all methods ofidentifying glycoproteins known in the art. These methods include, butare not limited to, immunohistochemistry techniques such asimmunoblotting or Western blotting, immunoperoxidase staining,fluorescein labeling, diaminobenzadine and biotinylation.

[0032] Generally, immunohistochemistry involves staining cryosectionedtissue samples. As used herein, endometrium samples may be cryosectionedto about 4-8μ thick. Endometrium is contacted with primary antibody,such as SSA6, followed by contact with secondary antibody, such asbiotinylated goat anti-mouse antibody. Endometrium is then incubated inavidin-conjugated horseradish peroxidase macromolecular complex followedby chromagen incubation, such as diaminobenzadine. Fluorescein may thenbe added to observe integrin distribution.

[0033] Immunoblotting involves the analysis of protein, here integrin,on sodium dodecylsulfate-polyacrylamide gel electrophoresis SDS-PAGE.The gel is run under monreducing conditions and the samples aretransferred to a nitrocellulose membrane for example. The membrane isincubated in media containing primary antibody, such as SSA6. The filteris developed using a secondary antibody, such as alkalinephosphatase-conjugated goat anti-mouse antibody.

[0034] The methods of diagnosing infertility and for detecting thewindow for embryo implantation in the endometrium of a mammal are alsowithin the scope of the invention. As provided herein, the β₃ subunit ofintegrin appears at day 20 of the menstrual cycle. It is also providedherein that α_(v)/β₃ on endometrial epithelium binds fibronectin,vitronectin and osteopontin. These molecules may provide a bridgebetween the α_(v)/β₃ integrin of the endometrium and the embryo.Further, patients with luteal phase dysfunction have delayed endometrialmaturation, infertility and negative staining for β₃ on days 20 through24. Thus, the optimal time for fertility may be determined byrepetitively testing endometrial samples at a plurality of stages in themenstrual cycle. As such, screening for β₃ provides a method ofdiagnosing infertility and for detecting the window of embryoimplantation in the endometrium. The window of implantation is that timewhen the endometrium of the uterus is available for embryo implantation.This window is preferably from day 19 to day 23, and more preferably day20 of the human menstrual cycle, marked by the expression of α_(v)/β₃integrin.

[0035] Similar cycles are known for other mammals—it is within theordinary skill in the art to adopt the foregoing methodology to suchcycles.

[0036] The present invention is also directed to methods of in vitrofertilization. Once the β₃ subunit of integrin is detected in an animalselected for pregnancy, a fertilizable egg (or eggs) from the same ordifferent animal could be replaced into the uterus to establishpregnancy. The egg and appropriate sperm are combined to produce azygote in vitro. For purposes of the invention, in vitro fertilizationmay take place in a petri dish, in a test tube or the like. In addition,in vitro fertilization may also refer to independently adding eggs andsperm to the fallopian tubes such that the zygote is formed therein. Inany event, the zygote is introduced to the uterus of the animal selectedfor pregnancy and monitored for implantation into the endometrium of theuterine wall.

[0037] Alternatively, the invention is directed to methods of preventingembryo implantation. Such may be carried out by contacting theendometrium with a neutralizing Fab fragment specific for β₃. Forpurposes of the present invention, Fab fragments from monoclonalantibodies which bind β₃ are within the scope of the invention. Fabfragments include, but are not limited to, Fab fragments from monoclonalantibodies SSA6 and 23C6. The Fab fragment may remain in vivo for atherapeutically effective time to prevent embryo implantation. The Fabfragment comprises the ligand binding portion of a monoclonal antibodyfor β₃, i.e. the binding site for β₃. A neutralized Fab fragment is usedin place of a typical monoclonal antibody to reduce the possibility ofan inflammatory reaction.

[0038] Contraception is a further embodiment of the invention. Acontraceptive may include a therapeutically effective amount ofneutralizing Fab fragment monoclonal antibodies specific for β₃ in apharmaceutically acceptable carrier, preferably adapted for intrauterineapplication.

[0039] Compounds which specifically block binding of the embryo to thisα_(v)/β₃ receptor are also included within the scope of the presentinvention. Examples include peptides containing the amino acid sequencearginine-glycine-aspartic acid, RGD (Pierschbacher et al., “SyntheticPeptide with Cell Attachment Activity of Fibronectin”, PNAS, Vol. 80,1224-1227 (1983)) which is the active binding site for the vitronectinreceptor. This sequence has been reported to block attachment ofpregnancy derived cells (trophoblast) in vitro by researchers, Kao etal., “The Human Villous Cytotrophoblast: Interactions with ExtracellularMatrix Proteins, Endocrine Function, and Cytoplasmic Differentiatien inthe Absence of Syncytium Formation”, Development, Vol. 130, 693-702(1988). Thus, a contraceptive containing the sequence RGD may beadministered locally to prevent embryo implantation.

[0040] Pharmaceutically acceptable carriers include, and are not limitedto, vaginal suppositories, intrauterine devices (IUD), gels such as slowrelease formulation, for example, depo forms of hormones—microcrystalsinjected and slowly released into the systemic circulation or deliveredin silastic tubing. Contraceptive formulations would be administered inabout 10 μg/ml.

[0041] Methods of monitoring endometrial maturation is also within thescope of the present invention. The endometrium may be monitored forembryo receptivity, embryo implantation, infertility, endometrialreplenishment and ovulation.

[0042] Diagnostic kits are also within the scope of this invention. Suchkits include monoclonal antibodies to rapidly detect β₃ in solution; anabsorbant detection device which contains pre-absorbed antibody againstβ₃ and to which uterine washings can be applied; a developer to make β₃visible when present.

[0043] The present invention is further described in the followingexamples. These examples are not to be construed as limiting the scopeof the appended claims.

EXAMPLES

[0044] Human Samples

[0045] Endometrium was obtained from 35 reproductive age women at thetime of hysterectomy. Tissue was obtained from the early proliferative(day 5) through late secretory phase (day 28) and all hysterectomieswere performed for benign disease. Endometrial biopsies were performedon women as part of their evaluation for infertility. All patients werecycling normally and none had received hormones for at least 3 monthsprior to surgery. Dating of the endometrium was assessed according tothe criteria of Noyes et al., “Dating the Endometrium” Fertil. Steril.,1:3-8 (1950). Endometrial biopsies were evaluated in the context oftiming of ovulation and/or the onset of the next menstrual period.Samples were judged as “out of phase” if histologic dating was delayedby 3 or more days relative to the predicted day of the menstrual cycle.Proliferative endometrium was categorized based on histology and on lastmenstrual period. Samples were transported on ice to the laboratory andwere snap frozen on dry ice and stored at −70° C.

[0046] Antibodies

[0047] Monoclonal antibodies (Mabs) PIH5, PIB5, PID6 specific to α₂, α₃,α₅ subunits, respectively, were acquired from Drs. Elizabeth Wayner andWilliam Carter. Mabs TS2/7 and B-5H10 directed against the α₁ and α₄subunits, respectively were acquired from Dr. Martin Hemler. GoH3, aspecific Mab directed against α₆ was acquired from Dr. ArnoudSonnenberg. Mab SSA6 specific to the β₃ subunit was acquired from Drs.Joel Bennett and James Hoxie. Mab LM142 against α_(v) was acquired fromDr. David Cheresh. The β₄ antibody was acquired from Dr. Steven Kennel.The 23C6 antibody which recognizes β₃ attached to α_(v) was obtainedfrom Michael Horton.

[0048] Immunohistochemistry

[0049] Immunoperoxidase staining was performed on cryostat sections ofendometrium samples from throughout the menstrual cycle. Serialcryosections 4-8μ thick were placed onto poly-L lysine coated slides,fixed in −20° C. acetone for 10 minutes, and stained using VectastainElite® ABC kits (Vector Laboratories, Burlingame, Calif.).Diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, Mo.) was used asthe chromagen. Primary antibody was placed on cryosections followingblocking with 1% bovine serum albumin in PBS, and allowed to bind atroom temperature for 1 hour. A phosphate buffered saline (PBS) pH 7.2 to7.4 rinse was followed by secondary antibody consisting of biotinylatedgoat anti-mouse antibody for 30 minutes. Following a PBS rinse, theendogenous peroxidases were quenched with a 30 minute incubation with0.3% H₂O₂ in absolute ethanol, followed by a 30 minute rehydration inPBS. Avidin:biotinylated horseradish peroxidase macromolecular complex(ABC) was then incubated on the sections for 30 minutes before addingdiaminobenzadine for 3 minutes to complete the reaction. Some sampleswere treated with 1:200 dilution of fluorescein-labeled anti-mouseantibodies for 1 hr., for immunofluorescent microscopy. Samples weresubsequently washed in PBS and mounted. The resulting staining wasevaluated on a Nikon microscope at low (100×) and higher (400×)magnification with or without fluorescence. Staining was judged asabsent (−), weak (±), moderate (+) or strong (++). Examples of each ispresented in FIG. 3. Photomicrographs were made using Kodak T-MAX 100ASA film.

[0050] Integrin Distribution in Normal Endothelium

[0051] The distribution of α₂, α₃, α₆, and β₄ subunits of integrinswhich recognize primarily collagen (Col) and/or laminin (LM) is shown inFIG. 1 A-D. These subunits were present on glandular epithelium (←)throughout the menstrual cycle. Their distribution within theendometrium was typical of that seen for most epithelial tissues. The α₂and α₃ subunits were distributed around the entire circumference of thecells, while the α₆ and β₄ subunits appeared to be localized at thebasolateral surface, adjacent to the basement membrane (BM) of theendometrial glands. The expression of these subunits by the mesenchyme(*) was less pronounced. While moderate staining was seen for α₆ onstromal cells (FIG. 1C) very little staining was noted for β₄. Theexpression of the α₄ and α₅ subunits of integrins known to bindfibronectin (Table 1) was quite restricted. The α₄ subunit wasundetectable above background staining (compare FIG. 1E with FIG. 2C) ineither epithelium or mesenchyme. The α₅ subunit (FIG. 1F),representative of the classic fibronectin receptor was not seen on theepithelial components, but was strongly expressed in the mesenchynewhich is rich in fibronectin. TABLE 1 DISTRIBUTION OF INTEGRINS BYLIGAND SPECIFICITY Ligands Integrin subunit Collagen/LamininFibronectin/Vitronectin α₁/β₁ α₄/β₁ α₃/β₁ α_(v)/β₁ α₂/β₁ α₅/β₁ α₆/β₁α_(v)/β₃ α₆/β₁ α₆/β₄

[0052] The intensity of immunostaining for three other subunits ofintegrins was found to change in a menstrual cycle-dependent manner.Immunostaining for α₁ in the proliferative phase (FIG. 2A) was onlyslightly above background levels (FIG. 2C). The intensity of stainingincreased throughout the secretory phase (FIG. 2B). This intensecircumferential staining was found on glandular and luminal epitheliumon all samples from day 15 to 28. Likewise, α_(v) was weakly expressedon both the epithelium and mesenchyme in the proliferative phase (FIG.3A) and staining increased gradually during the secretory phase to thelevel noted in FIG. 3B. During the proliferative phase β₃ staining wasonly present on the mesenchymal cells (FIG. 3C). Increased β₃ stainingwas apparent on the endometrial epithelium only after day 19 of themenstrual cycle (FIG. 3D) on the luminal as well as glandularepithelium, and was also present in a pericellular distribution. Incontrast, the basalis layer did not significantly stain for either α_(v)or β₃. This changing pattern of epithelial α_(v) and β₃ throughout thecycle was studied in 35 endometrial samples and is depicted graphicallyin FIG. 4 A and B.

[0053] Collagen/laminin receptors (col/LM) characterized by α₂, α₃ andα₆ were uniformly expressed throughout the menstrual cycle, see Table 2.The pericellular distribution of α₂ and α₃ subunits was distinctlydifferent from that of α₆ subunit. Characteristic of a laminin receptor,α₆ was concentrated on the basolateral surface. The β₄ subunit whichpairs with α₆, was also found distributed on the basolateral surface ofepithelial cells, and its distribution appeared even more restricted tothe basal pole. The α₅/β₁ integrin, a major fibronectin receptor, wasalso uniformly expressed throughout the menstrual cycle. Unlike thecollagen and laminin receptors, the distribution of α₅/β₁ was limited tothe mesenchyme.

[0054] The temporal pattern of distribution of α_(v) was varied.Immunostaining was first detected prior to the secretory phase with anincrease in intensity throughout the cycle. One subunit known to pairwith α_(v) is β₃. β₃ is not characteristically present on epithelialcells. The abrupt appearance of the β₃ subunit after day 19 suggeststhat expression of the vitronectin receptor is regulated in humanendometrium. The increased epithelial α_(v)/β₃ staining in normal cyclescorrelates to an implantation window within the secretory phase. Whilethe physiologic basis for the implantation window has not beenpreviously established, a proposed role of integrins in the initialinteraction between maternal and embryonic cells indicates anendometrial period of receptivity. TABLE 2 DISTRIBUTION OF INTEGRINSUBUNITS IN NORMAL ENDOMETRIUM DURING THE MENSTRUAL CYCLE Col/LM FN/VNCell Type α₁ α₂ α₃ α₆ β₄ α₄ α₅ α_(v) β₃ Epithelial ∘ • • •^(b) •^(b) ∘ ∘∘ ∘ proliferative early secretory • • • • • ∘ ∘ * ∘ late secretory • • •• • ∘ ∘ • • Stromal proliferative ∘ ∘ ∘ • ∘ ∘ • • • early secretory ∘ ∘∘ * ∘ ∘ * • • late secretory * ∘ ∘ • ∘ ∘ * • •

[0055] Integrins in Discordant Endometrium.

[0056] The presence of the epithelial β₃ subunit appeared to be aconsistent internal marker of luteal phase maturation, and the timing ofβ₃ expression correlated with the peri-implantation period or window ofembryo implantation. To investigate whether this phenomenon would beuseful in the clinical evaluation of endometrial biopsies,immunostaining was performed on luteal phase endometrial samples fromcycles which showed evidence of maturational delay. Endometrial biopsiesfrom 25 women who had concordance of menstrual and histologic dating(“Normal” group) were compared to 12 biopsies which were identified as≧3 days out of phase (OOP) based on either the time of ovulatory or thesubsequent menses. Samples were immunostained for α₁, α_(v) and β₃subunits. All biopsies were performed on days 20 to 24 of the menstrualcycle. In all instances, immunostaining for these three antigens waspresent on endometrial epithelia from the normal group. In biopsieswhich were delayed by 3 days or more, α₁ and α_(v) staining was present,but epithelial β₃ staining was absent. The comparison of β₃ stainingintensity in the two groups is shown in FIG. 5A. Accompanyingphotomicrographs of β₃ immunostaining from out of phase biopsies (OOP;B) and normal “in phase” biopsies (C) is included, which demonstratesthe discrepancy seen in β₃ staining. In subsequent treatment cycles, 2OOP patients underwent repeat biopsy during a normalized cycle at whichtime immunostaining for epithelial β₃ was present. This suggests thatthe lack of β₃ was not an intrinsic defect in the OOP group. Rather, thediscordant biopsies which lacked β₃ had not yet established themid-luteal phenotype of normal day 20 to 24 endometrium.

Cell Harvest and NP-40 Extraction

[0057] To further demonstrate that immunohistochemical stainingaccurately reflected changes in the expression of β₃ subunit onendometrial epithelium, immunoblots (Western blots) were performed onsamples of enriched endometrial glandular elements from proliferativeand secretory phase. Four samples of endometrium were obtained for theevaluation of the β₃ subunit in proliferative (n=2) and late secretory(n =2) endometrial epithelium. Each sample was placed in Dulbecco'smodified Eagle's medium (DMEM; Sigma, St. Louis, Mo.), supplemented with10% fetal bovine serum (Flow Laboratories, McLean, Va.) glucose (4500mg/L), Hepes buffer (25 mM), L-glutamine (584 mg/L), and sodiumbicarbonate (3.7 gms/L). Endometrium was minced in a plastic petri dishprior to incubation with 6 mg of collagenase (type 1A, 550 units/mg;Sigma, St Louis, Mo.) for 2 hours at 37° C. utilizing modifications ofthe procedures described by Satyaswaroop et al. in “Isolation andCulture of Human Endometrial Glands”, J Clin. Endocr. Metab., 48:639-641(1979). The resulting suspension was successively passed through a 250μm sieve and a 38 μm seive (Newark Wire Cloth Co, Newark N.J.). Thecourse (250 μm) sieve removed undigested material, while the secondretained the glandular elements and excluded the individual stromal andblood cells. After thorough rinsing, the glandular elements wereobtained by backwashing with 10 to 20 ml of DMEM. The isolated glandularstructures were then transferred to a 1.5 ml microfuge tube andcentrifuged 3 times (82×g) for 2 minutes with intermittent washes withPBS. Membrane extracts were prepared by adding small volumes (100-200μl) of 10 mM Tris-acetate, pH 8.0, 0.5% NP-40, 0.5 mM Ca²⁺ (TNC) with 2mM PMSF (phenyl methyl-sulfonyl fluoride) to the final pellet, pipettedand incubated on ice for 15 minutes. The lysate was centrifuged for 5minutes at 16,000×g in a microcentrifuge. The resulting supernatant wascalled NP-40 extract and was frozen at −70° C. until use. A portion ofthe original, undigested tissue was cryosectioned forimmunohistochemical localization of β₃.

[0058] Gel Electrophoresis and Immunoblots

[0059] The protein concentration of each NP-40 extract and an extract ofplatelets (positive control) was determined using technique described byLowry et al., “Protein Measurement with Folin Phenol Reagent”, J. Biol.Chem., 193:265-271 (1951). Samples with equal amounts of protein wereadded to electrophoresis sample buffer (62.5 mM Tris base, 2% SDS, 10%glycerol, pH 6.8). Samples were analyzed by SDS-PAGE using 6%polyacrylamide gels, using non-reducing conditions described by Laemmli,U.K., “Cleavage of Structural Proteins During Assembly of the Head ofBacteriophage T4”, Nature, 227:680-685 (1970). The gel was transferredto nitrocellulose using a Biorad transfer apparatus (Biorad, Richmond,Calif.) and blocked with 4% BSA in PBS with 0.2% Na Azide for 1 hour.After addition of primary antibody (SSA6 supernatant) for 2 hours, thegels were developed using an alkaline phosphatase-conjugated secondaryantibody (Promega Corp., Madison, Wis.) according to methods describedby Albelda et al., “EndoCAM:A Novel Endothelial Cell-Cell AdhesionMolecule”, J. Cell Biol., 1 10:1227-1237 (1990).

[0060] As shown in FIG. 6A, proliferative phase epithelial structureshad little to no immunostaining at 95 kD (lanes 2 and 3), compared tothe positive control (platelet extract; lane 1) or to samples from thesecretory phase (lanes 4 and 5) which showed strong staining for β₃. Theisolated endometrial glands appeared as tubular structures free ofsurrounding stroma (FIG. 6B). Immunofluorescent staining for β₃ fromsamples corresponding to lanes 3 and 4 (mid proliferative phase and day23, respectively) are shown in FIGS. 6C and D. Note the absence ofglandular staining in the proliferative sample, while both glandular andluminal immunostaining is obvious from the secretory phase. These dataconfirm that the expression of epithelial β₃ in human endometrium is acycle specific phenomenon.

[0061] Various modifications of the invention in addition to those shownand described herein will be apparent to those skilled in the art fromthe foregoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

1. A method for detecting receptivity of the endometrium of a mammal toembryo implantation, comprising the steps of: a) detecting β₃ integrinsubunit in an endometrial sample from the mammal; and b) correlating thepresence of the β₃ integrin subunit with endometrial receptivity.
 2. Themethod of claim 1, wherein the sample of endometrium is obtainedsurgically.
 3. The method of claim 2, wherein the sample is obtained bya biopsy or by a dilation and curettage.
 4. The method of claim 1,wherein the sample of endometrium is obtained nonsurgically.
 5. Themethod of claim 4, wherein the sample is obtained by a uterine washingor by a uterine brushing.
 6. The method of claim 1, wherein the β₃subunit is combined with another integrin subunit.
 7. A method fordiagnosing infertility in a mammal, comprising the steps of: a)detecting the presence and level of a β₃ integrin subunit in anendometrium sample of a mammal at a plurality of stages of theendometrial cycle; and b) correlating delayed appearance or a reducedlevel of β₃ integrin subunit expression with infertility.
 8. The methodof claim 7, wherein expression is monitored during a plurality of stagesof the endometrial cycle.
 9. The method of claim 7, wherein the sampleof endometrium is obtained surgically.
 10. The method of claim 9,wherein the sample is obtained by a biopsy or by a dilation andcurettage.
 11. The method of claim 7, wherein the sample of endometriumis obtained nonsurgically.
 12. The method of claim 11, wherein thesample is obtained by a uterine washing or by a uterine brushing. 13.The method of claim 7, wherein the β₃ subunit is combined with anotherintegrin subunit.
 14. A method of monitoring endometrial maturation in amammal, comprising: a) detecting the appearance of the β₃ subunit ofintegrin in a sample of endometrium obtained from the mammal at aplurality of stages of the endometrial cycle; and b) correlating theappearance of the β₃ subunit of integrin with endometrial maturation.15. The method of claim 14, wherein the appearance of β₃ is monitored ondays 19-23 of the menstrual cycle.
 16. The method of claim 14, whereinthe appearance of β₃ is monitored on days 20 to 24 of the menstrualcycle.
 17. The method of claim 14, wherein delayed maturation isindicative of infertility.
 18. The method of claim 14, wherein the β₃subunit is combined with another integrin subunit.
 19. A method ofdetecting the window of embryo implantation in endometrium in a mammal,comprising: a) detecting the presence of a β₃ integrin subunit inendometrium samples obtained from the mammal at a plurality of stages ofthe menstrual cycle; and b) correlating the presence of β₃ with thewindow of embryo implantation.
 20. The method of claim 19, wherein thesample of endometrium is obtained surgically.
 21. The method of claim20, wherein the sample is obtained by a biopsy or by a dilation andcurettage.
 22. The method of claim 19, wherein the sample of endometriumis obtained nonsurgically.